Project Summary Tumor heterogeneity is emerging as a major focus of cancer research. Tumors of many organs, including of the lung, exhibit intra-tumoral heterogeneity and different regions of a tumor can have varying resistance to therapies. A better understanding of tumor heterogeneity prior to treatment could be used for more personalized, informed treatments resulting in a better prognosis for patients. Formalin-fixed, paraffin-embedded (FFPE) sectioning of cancer specimens followed by cell staining is standard practice in pathological diagnosis. Unlike longer RNAs, microRNAs (miRNAs) can withstand the FFPE process and are known to be dysregulated in many diseases, especially cancer. Despite promise of miRNAs as sensitive and robust markers for cancer diagnosis and prognosis, there is no technology that can perform a quantitative, multiplexed detection of miRNA biomarkers from tumor tissue sections while preserving the spatial information of tumor biopsies and not being labor-intensive. The goal of this project is to develop tools to spatially and quantitatively resolve multiple miRNA biomarkers within pathological tissue sections. The proposed goals will be achieved via the following specific aims: 1) establishing a gel-post microwell platform for local miRNA measurement in small regions of tissue sections, 2) scaling up the sensing array and advancing the sample handling and data analysis, and 3) demonstrating the utility of the integrated system on patient-derived organoid and mouse xenograft samples, and genetically engineered mouse models. We have assembled a team of engineers and pathologists to achieve these goals. The technologies will be developed in a manner such that they integrate into and complement the current workflow of pathologists, thus providing a pathway to widespread adoption by clinicians. The project is innovative because no technology currently exists to perform spatially resolved miRNA expression profiles in clinically relevant FFPE tissue samples. The project is significant because it will provide researchers and pathologists a better understanding of tumor heterogeneity prior to treatment which could be used for more personalized, informed treatments resulting in a better prognosis for patients. The long-term goal is to include this assay in the standard of care tissue-based biomarker workup of every cancer patient.